1. Field of the Invention
This invention relates to the production of squalene for commercial and industrial uses. More particularly, the invention relates to a process by which natural squalene levels in plants can be increased, and to nucleotide sequences that can be introduced into plants to cause the desired increase, and plasmids, vectors, etc., useful in the process.
2. Description of the Related Art
There is a U.S. $125 million per annum market for squalene, a colourless oil used in the cosmetics and health industries (Kaiya, 1990). Squalene is currently obtained mainly from shark liver, but it also occurs in small quantities in vegetable oils. Squalene extracted from shark liver is declining in supply (Kaiya 1990) and the harvesting of sharks for this purpose is anyway environmentally unfriendly and is becoming less acceptable as environmental concerns increase in society.
Squalene can be extracted from olive oil, although the amounts are not sufficient to supply even the cosmetics market (Bondioli et al. 1992; Bondioli et al. 1993). Squalene could be extracted from other vegetable oils, but the levels of the hydrocarbon in the oil are too low for this to be economically viable. There are at present no Canadian crops used for squalene production. It has been suggested that, if the levels of squalene occurring in oilseeds could be increased, the traditional source of squalene could be replaced by oilseed crops, to the benefit of both the environment and those countries, such as Canada, that grow crops of this kind in abundance. Many vegetable oils undergo deodorization by vacuum distillation as a routine part of refining. Most of the squalene in the oil can be recovered in the deodorizer distillate which is a by-product of this process (Bondioli et al., 1993). Typically, squalene is concentrated more than one hundred fold in the deodorizer distillate relative to the levels in unrefined vegetable oils. For commercial viability, vegetable oil deodorizer distillates should contain at least 5% (w/w) squalene. Currently, soybean and canola deodorizer distillates contain squalene in the 0.1-3% range (Ramamurthi, S., 1994). Consequently, an increase of two-fold or more in the squalene content of these oilseeds could result in commercially viable squalene production from vegetable oils.
It has been shown that in plant cell cultures, squalene accumulates in the presence of squalene epoxidase inhibitors, e.g. allylamines such as terbinafine (Yates et al. 1991). Apparently, much of the squalene produced in plants is converted to the epoxide by squalene epoxidase, and ultimately to plant sterols. In fact, all plant and higher life forms contain squalene and squalene epoxidase genes, but little squalene accumulates in the tissues of such life forms because of the effects of the expressed squalene epoxidase. Therefore, inhibition of the epoxidase gives squalene an opportunity to accumulate. However, there are as yet no commercial processes based on this concept.
A main problem addressed by the inventors of the present invention is therefore to create a plant crop, particularly an oilseed crop, which accumulates squalene in harvestable tissues, such as seeds, at sufficient levels for commercially-viable extraction.